This proposal is focussed on the structure - function relationships of the pyruvate dehydrogenase isolated from E. coli. In particular, crosslinking studies of the complex are designed to determine the site of protein-protein contact among the 72 subunits of the complex. This will be done by crosslinking the complex using a water soluble carbodiimide and, subsequently, comparing tryptic mapping of the crosslinked complex (or its sub- complex) with that of the untreated complex (or subcomplex). Double labelled complexes will also be prepared by growing cells on 14C or 3H media, isolating the complex, resolving them and reconstituting them with one component 14C labelled, the other 3H labelled. These complexes will be crosslinked and the crosslinked material tryptically mapped to find the double labelled peptide which, of course, had its origin at the protein- protein junction between the two different (E1, E2, or E3) subunits. Secondly, specific crosslinking studies will be undertaken to determine what amino acid residues, and on which subunit, are contacted by the lipoyl mority. This will be done using materials that will label the lipoyl group specifically under initial conditions and, subsequently, can be caused to crosslink with any nearby residue. Finally, if time permits, the mobility of the lipoyl group will be investigated using 13C NMR. In these investigations, cells requiring lipoic acid will be grown on 13C enriched lipoate and the complex will be isolated from the cells. The NMR of the resulting complex will be determined in the presence and absence of substrates. By comparing the 13C NMR line widths we can tell if any substrate, or combination of substrates, trigger the movement of the lipoyl group.